Hepatitis C virus (HCV) infection represents a significant global health-care problem, which is forecasted to become worse in the coming years. In the developed world, infection with HCV is responsible for 50-75% of all cases of liver cancer and accounts for two-thirds of all liver transplants. To date there are no effective vaccines to HCV and current systemic therapies have significant side effects. There is a need for novel therapeutic approaches to the treatment of chronic hepatitis C infection. The establishment of potent anti-viral CD8+ T-cell immunity has been shown to be the central mediator of viral clearance. Like many chronic infections however, such responses to HCV have been difficult to establish. Plasmacytoid dendritic cells (pDCs) are a subset of DCs which are specialized for viral recognition and the initiation of anti-viral immunity. We have shown that pDCs have specialized antigen processing compartments (MICs) which permit them to rapidly cross-present viral antigens and stimulate protective CD8+ T-cell responses. Furthermore we have demonstrated that targeting antigens to this compartment in an activated pDC is sufficient to initiate potent CD8+ T-cell responses. Our overall hypothesis is that Hepatitis C viral antigens targeted to the specialized class-I processing compartment (MIC) of pDCs will be efficiently cross-presented and drive anti-viral CD8+ T cell expansion. We propose to address this hypothesis though three aims;Aim 1: To determine if receptor trafficking into the MIC is sufficient to generate strong CD8+ T-cell responses against Hepatitis C viral antigens. We will address this hypothesis by 1) Generating antibody antigen conjugates for in vitro targeting to the MIC. 2) Assess the effect of these reagents on pDC activation 3) Demonstrate MIC targeting 4) Demonstrate cross- presentation of targeted antigen. Aim 2: To determine if antigen processing and cross-presentation by the pDC results in an expanded antigen specific T-cell repertoire. We will 1) Demonstrate that antibody antigen conjugates can induce potent HCV antigen specific CD8+ T cells responses in vitro 2) Determine the optimal CpG derivative (CICs) to enhance viral antigen specific CD8+ T cell responses 3) Make both a quantitative and qualitative assessment of total T-cell epitopes generated during cross-presentation of targeted viral antigens by pDCs. 4) Assess the quality of viral epitope response in patients chronically infected with HCV. Aim 3: The generation of a multi-epitopic adjuvant-based pDC targeting constructs We will 1) Generate fusion proteins of anti-BDCA2 and the immunodominant TC1 viral epitopes identified in Aim 2. 2) Conjugate immunostimulatory CIC sequences to this second generation pDC targeting construct. 3) Demonstrate that a multi-epitopic pDC targeting constructs can induce potent HCV antigen specific CD8+ T cells responses in vitro in patients chronically infected with HCV. Overall significance: This study provides a novel approach for therapeutic HCV vaccine development. PUBLIC HEALTH RELEVANCE: Overall this study provides a novel approach for HCV therapeutic vaccine development. These studies will demonstrate for the first time that targeting antigen and adjuvant to a specific compartment within pDCs induces potent CD8+ T-cell immunity against HCV antigens, and thus permits the development of novel, rationally designed, vaccines.